In Saccharomyces cerevisiae, the SLN1, YPD1, and SSK1 proteins form a multi-step Hjs-Asp phosphorelay signaling pathway that controls adaptive responses to hyperosmotic stress. YPD1, a prototypical histidine-containing phosphotransfer (HPt) protein, is required for phosphoryl group transfer from the membrane-bound sensor histidine kinase SLN1 to the response regulator protein SSK1. The focus of this proposal is on the regulation of SSK1 function by phosphorylation and dephosphorylation. YPD1 serves a dual function in the yeast osmoregulatory pathway by shuttling phosphoryl groups to SSK1 and also by stabilizing the phosphorylated state of SSK1 under non-osmotic stress conditions. Contrary to most two-component response regulator proteins, SSK1 is rendered inactive when phosphorylated. Hyperosmotic stress, through a mechanism that is poorly understood, results in rapid dephosphorylation of SSK1, which allows SSK1 to interact with and activate a downstream MAP kinase cascade. Very little is known about how environmental conditions influence the association or dissociation of regulatory protein complexes. Hence, the studies proposed herein will address how YPD1/SSK1 interactions are influenced by both environmental conditions and the response regulator phosphorylation state. Mechanism(s) by which SSK1 is rapidly dephosphorylated in response to hyperosmotic stress will also be examined. The specific aims of this application are to i) obtain co-crystal structures of YPD1-response regulator complexes, ii) examine environmental conditions that affect YPD1/SSK1 complex formation both in vivo and in vitro, and iii) examine possible mechanism(s) of activation of SSK1 via dephosphorylation. This work has significance to two major areas of basic scientific and biomedical importance. Foremost, the proposed studies will lead to a better understanding of regulated protein-protein interactions in the context of cell signaling pathways. In a broader context, these studies may provide a basis for development of novel antibacterial and antifungal drugs.